DESCRIPTION (from applicant's Abstract) The proper intracellular folding and trafficking of membrane proteins, including transporters, channels, and receptors are key events that contribuute to their function. Ste6p, the mating pheromone a-factor transporter in yeast, is being used as a model protein for dissecting the trafficking steps of mulitspanning membrane proteins. Ste6p is a member of the ATP binding cassette (ABC) superfamily that includes the cystic fibrosis protein CFTR, multidrug resistance transporters, and related proteins important in human health and disease. Studies proposed here focus on two aspects of Ste6p trafficking that represent key regulatory points for a plasma membrane protein: 1) ER quality control, a process intertwined with ER exit, and 2) endocytosis. The applicant cites several advances in the previous project period: a) The identification of distinct classes of ER-retained ste6 mutants, which revealed considerable complexity in ER quality control; b) Discovery of a prominent novel structure, the ER-associated body (ERAB) that is formed in response to certain ste6 mutants and may provide clues about ER quality control; c) Demonstration of a role for ubiquitin both in ER quality control and endocytosis of Ste6p. These findings set the stage for the present goal of identifying cellular components involved in "decision making" aspects of ER exit and endocytosis. The renewal lists the following aims: 1) Isolate chromosomal suppressors and enhancers of ER-retained ste6 mutants to identify cellular components that participate in ER quality control and/or ER exit. 2) Identify cellular components enriched within ERABs or induced upon their formation, by biochemical, microscopic, and microarray analysis. 3) Establish by mutant studies an ordered pathway of events (phosphorylation -> ubiquitination -> endocytosis (PUE pathway) required for Ste6p internalization, and genetically identify novel Ste6p endocytosis specficity factors. 4) Compare the requirements for ubqiuitination leading to ER-associated degradation (ERAD) of mutant Ste6p versus endocytosis of WT Ste6p. Human membrane protein "trafficking diseases" are numerous. The applicant hopes that these efforts to identify cellular components involved in trafficking will provide targets for rational drug design for these and related diseases.